Ziska S E, Bhattacharjee M, Herber R L, Qasba P K, Vonderhaar B K
Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892.
Endocrinology. 1988 Nov;123(5):2242-8. doi: 10.1210/endo-123-5-2242.
Mouse mammary tissue, when cultured in the presence of insulin, corticoids, PRL, and physiological levels of T3, shows increased synthesis and secretion of alpha-lactalbumin. Tissue cultured in the presence of insulin, hydrocortisone, PRL, and T3 synthesizes two distinct forms of alpha-lactalbumin, but secretes only one form. Tissue cultured in the absence of T3 synthesizes and secretes only one form. To address the question of whether these two electrophoretically distinct forms arose by differential glycosylation of the same polypeptide or by synthesis of two different polypeptide precursor chains, mammary tissue was cultured in the presence of insulin, corticoids, and PRL with or without T3, and the mRNA and alpha-lactalbumins were isolated. Northern blot analyses indicated that mammary gland tissue cultured in the presence of T3 contained 2.46 times more alpha-lactalbumin mRNA than tissue cultured only in the presence of insulin, hydrocortisone, and PRL. This enhanced mRNA level was confirmed by in vitro translation experiments where tissue cultured in the presence of insulin, hydrocortisone, PRL, and T3 produced mRNA that resulted in 2.1 times as much radiolabeled alpha-lactalbumin as tissue cultured in the absence of T3. Sodium dodecyl sulfate-polyacrylamide gel analysis of the in vitro translation products revealed only one band, suggesting the presence of only one message. Endoglycosidase digestion of the two forms of alpha-lactalbumin produced in the presence of T3 resolved them into a single band on sodium dodecyl sulfate-polyacrylamide gels. Thus, the electrophoretic differences between the two forms synthesized in the presence of T3 appear to be due to differential N-linked glycosylation of the same polypeptide chain and not to synthesis of two different polypeptide precursor chains.
小鼠乳腺组织在胰岛素、皮质类固醇、催乳素（PRL）和生理水平的三碘甲状腺原氨酸（T3）存在的情况下进行培养时，α-乳白蛋白的合成和分泌会增加。在胰岛素、氢化可的松、PRL和T3存在的情况下培养的组织会合成两种不同形式的α-乳白蛋白，但只分泌一种形式。在没有T3的情况下培养的组织只合成和分泌一种形式。为了解决这两种电泳上不同的形式是由同一多肽的差异糖基化还是由两种不同的多肽前体链的合成产生的问题，乳腺组织在有或没有T3的胰岛素、皮质类固醇和PRL存在的情况下进行培养，并分离出mRNA和α-乳白蛋白。Northern印迹分析表明，在T3存在的情况下培养的乳腺组织中α-乳白蛋白mRNA的含量比仅在胰岛素、氢化可的松和PRL存在的情况下培养的组织多2.46倍。这种mRNA水平的提高在体外翻译实验中得到了证实，在该实验中，在胰岛素、氢化可的松、PRL和T3存在的情况下培养的组织产生的mRNA导致放射性标记的α-乳白蛋白是在没有T3的情况下培养的组织的2.1倍。对体外翻译产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶分析只显示出一条带，表明只存在一种信息。对在T3存在的情况下产生的两种形式的α-乳白蛋白进行内切糖苷酶消化后，在十二烷基硫酸钠-聚丙烯酰胺凝胶上它们被分解成一条带。因此，在T3存在的情况下合成的两种形式之间的电泳差异似乎是由于同一多肽链的差异N-连接糖基化，而不是由于两种不同的多肽前体链的合成。
